Ampicillin-chloral hydrate complex

ABSTRACT

Novel 6-(D-2-amino-2-phenylacetamido)penicillanic acid-2,2,2trichloro-1,1-ethanediol complex. The complex is useful as a broad spectrum antibiotic.

United States Patent Higuchi et al.

AMPlCILLIN-CHLORAL HYDRATE COMPLEX Inventors: Takeru Higuchi; Kaneto Uekama,

both of Lawrence, Kans.

U.S. Cl 260/239.1; 424/27l Int. Cl C07d 99/16 Field of Search 260/239.1

References Cited UNITED STATES PATENTS 5/i96l Doyle et a]. 260/239i (451 June 10, 1975 3,198,788 8/1965 Granatek et 260/2391 OTHER PUBLICATIONS Fieser et al., Organic Chemistry, pp. 202-203 (1950). The Merck Index, p. 232, (Eighth Edition), [968.

Primary Examiner-Nicholas S. Rizzo Attorney, Agent, or FirmPaul L. Sabatine; Steven D. Goldby; Edward L. Mandel! [57] ABSTRACT Novel 6-(D-Z-amino-Z-phenylacetarnido)penicillanic acid-2,2,2-trichloro-l,l-ethanedioi complex. The complex is useful as a broad spectrum antibiotic.

1 Claim, 11 Drawing Figures SPECIFIC ROTATION (DEGREES X I6 PATENTEUJUN 10 ms 3 888,848

SHEET WAVELENGTH mm DEGREES Fig.4

TIME (SECONDS) INVENTORS Takeru Higuchi Kaneto Uekama W A a(. M

Attorneys PATENTEDJUH 10 ms 3 s5 g SHEET 3 WAVENUMBER m5 F i g. 3

8' Q s 9 a P1 BONVLLIWBONVHL INVENTORS Takeru Higuchi BY Kanefo Uekama Attorneys PATENTEIJJUH 1 0 ms 11% 888,848

SHEET 4 MOLAR CONCENTRATION OF AMPICILLIN X IO 4:.

I I I T 0 IO 20 3O 40 F i g 5 MOLAR CONCENTRATION OF LIGAND x10 2'0 2 INVENTORS F i g 6 MOLAR CONCENTRATION OF LIGAND x IO Takeru Hiquchi BY Kanero Uekama Attorneys PATENTEDJUH I 0 I975 CONCENTRATION OF AMPICILLIN (MG/ML) 3.8881348 SHEET 5 TIME (SEC x I62) INVENTORS Takeru Hiquchi BY Kanero Uekama Attorneys PATENTEDJUH I 0 ms .8 8

SHEET 6 TIME (SEC x I62) Fig.8

INVENTORS Takeru Higuchi BY Kaneto Uekama Attorneys AMPICILLIN-CHLORAL HYDRATE COMPLEX BACKGROUND OF THE INVENTION The present invention pertains to both novel and use ful ampicillin-chloral hydrate complex. More particularly, the invention relates to the product 6-(D-2- amino-2-phenylacetamido)penicillanic acid-2.2,2-trichloro-l,l-ethanediol, or ampicillin-chloral hydrate complex, that possesses valuable broad spectrum antibacterial activity.

The pharmaceutically important compound 6-(D-2- amino-2-phenylacetamido)penicillanic acid, also known by the generic term ampicillin is of proven therapeutic value as a broad spectrum antibacterial agent. Ampicillin is therapeutically useful in mammals, including man, avians, farm animals. household and sport animals for the management of infectious diseases caused by gram-positive and gram-negative bacteria and also because it is well absorbed from the gastrointestinal tract. Additionally. ampicillin has value as a nutritional supplement in animal feeds.

Ampicillin, as disclosed in U.S. Patent No. 2,985,648, is presently used for the above therapeutic and nutritional purposes in two different forms. One form is comprised of crystalline D-6-(2-amino-2 phenylacetamido)penicillanic acid, commonly known as anhydrous ampillicin, as described in US. Pat. Nos. 3,144,445 and 3,299,046, and the other form comprised of 6-( D-2-amino-2- phenylacetamido)penicillanic acid trihydrate, commonly known as ampicillin trihydrate and described in US. Pat. Nos. 3,157,640 and 3,299,046. Although ampicillin, in both forms, is widely used for its proven activities, the biological activity of these ampicillins after administration to a host is often delayed, or it reaches a desired peak only after a prolonged period of time. Generally, this is attributed to the poor or slow bioavailability of the anhydrous and trihydrate forms of ampicillin from the administered dosage form or as pure drug, which bioavailability is dependent on the rate of dissolution of the two forms of ampicillin from pharmaceutical formulations containing the respective forms or the forms per se. That is, where the rate of dissolution of the administered dosage formulation comprised of anhydrous ampicillin or ampicillin trihydrate is low or it is characterized by a prolonged rate of dissolution, the rate of dissolution becomes the rate-limiting step in the pharmacodynamic processes controlling the bioavailability of ampicillin.

SUMMARY OF THE INVENTION Accordingly, it is an immediate object of this invention to provide a novel and useful form of penicillin that substantially overcomes the difficulties associated with the prior art forms of ampicillin.

Yet another purpose of this invention is to provide a new form of ampicillin with improved bioavailability by making available a form of ampicillin that possesses an increased rate of dissolution.

Still another purpose of the invention is to provide a new form of ampicillin that is relatively easy to make and avoids the needs of expensive precursors.

Yet still anoter purpose of the invention is to provide a new ampicillin having an increased rate of dissolution and synthesized as the reaction product of ampicillin and chloral hydrate.

In accomplishing these objects, and other features and advantages, this invention makes available to the art a new and useful form of ampicillin, 6-(D-2-amino- 2-phenylacetamido)penicillanic acid-2,2,2-trichloro 1,1-ethanediol complex having an improved rate of dissolution while retaining the valuable broad spectrum activity of the parent ampicillin.

Other objects, features and advantages of this invention will become more apparent from the following detailed description of the invention and the accompany ing claims.

DETAILED DESCRIPTION OF THE INVENTION The novel and useful product of the invention. 6-[D- 2-amino-Z-phenylacetamido)penicillanic acid2.2,2- trichloro-1,1-ethanediol complex, referred to herein as ampicillin-chloral hydrate complex, can be synthesized by intimately contacting and reacting 6-(D-2-amino-2- phenylacetamido)penicillanic acid, ampicillin, with 2,- 2,2-trichloro-1,1-ethanediol, chloral hydrate. Generally, the reaction is carried out by reacting 1 mole or an excess thereof of anhydrous ampicillin with 2 moles or an excess thereof of chloral hydrate at a temperature of 15C to 60C, at atmospheric pressure for about 30 minutes to about 4 hours in the presences of an aqueous solvent or the like. The product is recovered from the reaction medium by first cooling the reactants, then filtering the precipitate, and finally drying it under reduced pressure to yield fast dissolving ampicillinchloral hydrate complex.

The following example will serve to illustrate the synthesis of ampicillin-chloral hydrate complex, without limiting the invention thereto.

EXAMPLE I To 30 grams of 2,2,2-trichloro-l,l-ethanediol, chloral hydrate, in a milliliter borosilicate Erlenmeyer flask is added 5 grams of anhydrous 6-(D-2-amino-2- phenylacetamido)penicillanic acid, anhydrous ampicillin, and the flask agitated to mix the compounds. Next, 50 milliliters of freshly prepared. double distilled water is added to the flask and the flask again agitated to mix the reactants. The flask is then heated to 40C at atmospheric pressure and it is maintained at these conditions for about 1 hour with the flask continuously agitated during this period. At the end of the period, the flask is cooled by placing it in a conventional dry iceacetone bath to precipitate the product, 6-( D-2-amino- Z-phenylacetamido)penicillanic acid-2,2,24richlorol,1-ethanediol complex, ampicillin-chloral hydrate complex. The precipitate is filtered, and dried in an oven at 25C under reduced pressure for about 20 hours. The recovered ampicillin-chloral hydrate complex is slightly yellowish, melted at 92-93C, and has a formula of C ,H ,N O,S.ZC H CI O v The elemental analysis for the complex is as follows: carbon calcu lated 35.28%, found 35.31%; hydrogen calculated 3.67%, found 3.88%; nitrogen calculated 6.17%, found 6.1 1%; sulfur calculated 4.60%, found 4.80%, and chlorine calculated 31.31%, found 29.96%.

The new ampicillin-chloral hydrate complex is further characterized by analyzing it by conventional physical and chemical techniques, and as shown by the accompanying Figures. In FIG. 1, the thermal behavior of ampicillin-chloral hydrate complex, curve C, is compared with ampicillin-trihydrate, curve T, and anhydrous ampicillin. An. The thermogram for anhydrous ampicillin shows a single exotherm at about 205C representing oxidative thermal degradation. The thermogram for ampicillin trihydrate shows an endotherm at l 10C and an exotherm at 208C, with the endotherm representing the dehydration of the material and the exotherm temperature indicating decomposition of its anhydrous form. The thermogram for ampicillin chloral hydrate shows an endotherm at 93C and an exotherm at l 14C. The endotherm at 93 represents melting of the complex.

AmpicilIin-chloral hydrate complex optical rotatory dispersion spectrum, RD. is illustrated in FIG. 2. In the figure, the ORD spectra of anhydrous ampicillin, An, is compared with ampicillin chloral hydrate complex, C. in a 0.1M phosphate buffer solution at pH 7.0. The maximum of both of the spectra is located at about 247 mp, with only small differences in the shorter wavelengths. In comparing the same amount of samples. the optical activity of ampicillin chloral hydrate complex is about half the activity of anhydrous ampicillin, indicating the composition of ampicillin-chloral hydrate complex to be l:2.

FIG. 3 is the partial infrared spectra in the 2000 cm to I400 cm region at 0.5% in KBr for anhydrous ampicillin, curve An, for ampicillin trihydrate, curve T, and for ampicillin chloral hydrate, curve C. with the spectrum of chloral hydrate shown as the dotted line. The spectrum of ampicillin trihydrate shows two strong bands, one centered at I780 cm due to the fused fidactam ring and the other at I690 cm due to the amide I and the characteristic of all penicillins. The bands between I650 cm and I500 cm represent the carbonyl stretchning band of the ionized carboxyl group, the amide II band and possibly NH deformation band. The infrared spectrum of ampicillin trihydrate differs from the infrared spectrum of anhydrous ampicillin in this region. The infrared spectrum of ampicillin-chloral hydrate complex produces bands for the B-lactam ring and for amide I which are identical with ampicillin, but the complexs bands are characterized by broadening and lowering frequency shift in the region between I650 cm and I500 cm".

FIG. 4 is a polarimetric recording at 247 mu obtained as a function of time for anhydrous ampicillin, curve An, and for ampicillin-chloral hydrate complex, curve C, as determined by the following spectropolarimetric penicillinase method. First, an aqueous solution of the ampicillin to be analyzed is prepared by dissolving 0.l gram of the respective ampicillin in I00 ml of 0.I M phosphate buffer solution at pH 7.0. Next, a I cm. cell is filled with this solution and placed in the spectropolarimeter. The assay is carried out at 247 mu with a spectral band width of 40 A a slit width of 2.4 mm, a chart speed of 25 sec/division, and 1.0 degrees. The rotation is recorded for I to 2 minutes. Then, ul of penicillinase solution having a potency of 80,000 units/ml is injected into the cell, the cell shaken, and the results recorded. In FIG. 4, the segment AB represents the optical rotatory activity prior to the addition of the enzyme. At B, penicillinase solution is injected, producing the rapid, linear decrease in rotation, segment C, corresponding to the enzymatic cleavage of the B-lactam ring to form a D-Obenzyl-penicilloate derivative, followed by a decrease in rotation in the terminal phase, segment D, due to mutarotation of D-O-benzyl-penicilloate. Ampicillin-chloral hydrate demonstrates the same results as ampicillin as seen by curve C.

The increased solubility of anhydrous ampicillin due to ampicillin chloral hydrate formation is seen in the phase diagram of FIG. 5. The solubility analysis is carried out as follows: first. an equal am of hydrous ampicillin is added into each of 20-ml screw cap vials, followed by increments of chloral hydrate. Next, the volume of each vial is brought to a final volume of IO ml with the selected solvent. and solubility data obtained at 25C for 48 hours or longer. Aliquots of the vials is removed and diluted with 0.1 M phosphate buffer, pH 7.0 and analyzed spectrophotometrically at 270 m;.;.. The results obtained as seen in FIG. 5 indicate an increased solubility of anhydrous ampicillin as the ampicillin-chloral hydrate complex forms, and that the increase continues until the apparent solubility limits of the complex is reached. In FIG. 6, the increased solubility of anhydrous ampicillin in the presence of the same lignand system is observed in ethyl acetate at 25C. FIG. 6 shows that the ampicillin-chloral hydrate complex is formed in non-aqueous solvents and that the solubilizing affect of chloral hydrate is stronger in the non-aqueous system than in the aqueous system. Similar results are obtained for dioxane and triacetin.

The dissolution rate curves for anhydrous ampicillin, curve An, for ampicillin trihydrate, curve T, and for ampicillin-chloral hydrate complex, curve C, are seen in FIG. 7. The dissolution rates are obtained by the tablet method as follows: first, 200 mg or 400 mg of vari ous forms of powdered ampicillin are compressed into tablets at a pressure of 5,000 lbs/sq. in., for 2 minutes in a Carver Laboratory Press, Model C. Next, ml of a solvent is added to a two neck round bottom flask, immersed in a constant temperature water bath, and a tablet added to the flask. The flask is agitated and aliquots drawn for spectrophotometric analysis at appropriate times. In FIG. 7, the dissolution rates are depicted for various forms of ampicillin at 25C in distilled water. The figure shows that the rate of dissolution for ampicillinchloral hydrate complex is greater than the rate of dissolution of anhydrous ampicillin or for ampicillin trihydrate. In FIG. 8. the rate of dissolution is illustrated for powdered samples of the three forms of ampicillin. The sampling procedures were as follows: first, 30 ml of solvent is added to a 50 ml Erlenmyer flask and then an excess of the sample is added thereto. The amount added is about 3 times the amount needed to saturate the solvent. Next, the solution in the flask is agitated and samples withdrawn periodically with cotton covered pipets and analyzed spectrophotometrically at 270 mu. FIG. 8 shows that the initial rate of dissolution for ampicillin-chloral hydrate is greater than the initial rate of dissolution for anhydrous ampicillin or ampicillin trihydrate with a corresponding availability of the complex.

The rate of dissolution in an acidic environment at a pH of 1.2 and temperature of 37C is illustrated in FIGS. 9, I0 and 11, for ampicillin-chloral hydrate complex, curve C, and for anhydrous ampicillin, curve An. The results of the comparison of anhydrous ampicilin and ampicillin-chloral hydrate complex as shown in FIG. 9 are by the tablet method, the results in FIG. 10 by the powder method, and in FIG. II by the capsule method. The capsule method is carried out by filling soft gellatin capsules with 500 mg of ampicillin, and then adding it to a 0. IN HCl-0.27r NaCl at pH 1.2, and

a temperature of 37C. At prescribed time intervals, samples were withdrawn with cotton covered pipets and spectrophotometrically analyzed at 262 mu. FIGS. 9, l0 and ll all show that the difference in dissolution rates between the complex in comparison with anhydrous ampicillin is still very significant. even in acid conditions.

The ampicillin-chloral hydrate complex is used for treating gram negative and gram positive infections in animals. including humans. farm animals, laboratory animals, household and sport animals. The oral dosage formulation is administered in a daily regimen of from 200 mg to gm. preferably about 4 to 6 times daily for gram negative infections and 2 to 4 times daily for gram positive infections. A typical tablet for oral dosage administration is represented by Example 2.

EXAMPLE 2 Ingredients Per Tablet. mg

Ampicillin-chloral hydrate complex calculated as ampicillin 251) Calcium carbonate I50 Cab-O-Sil i Sodium citrate, anhydrous Magnesium stearate 7.5 Amherlite XE 88 5 Microcel C 90 EXAMPLE 3 ingredients Per Capsule. mg

Ampicillin-chloral hydrate complex calculated as ampicilliu 21X] Calcium carbonate lUO Lactose. U.S.P. 21K] Starch I30 Magnesium Stearate 4.5

The above ingredients are blended together in a standard blender, and then discharged into commercially available capsules. When higher concentrations of the active agent are used, a corresponding reduction is made in the amount of lactose or capsules or tablet size is increased.

For administering the novel complex of the invention to valuable domestic household, sport or farm animals. such as dogs, sheep, goats, cattle, etc.. or for administering to laboratory animals such as mice. rats. guinea pigs. monkeys, etc.. for scientific studies. the complex can also be administered by preparing a food premix. such as mixing it with dried fish meal, oatmeal, straw. hay, ground corn. mash. and the like. and then the prepared premix is added to the regular feed, thereby administering the complex to the domestic or laboratory animal in the form of feed.

The above examples and disclosure are set forth merely for illustrating the mode and the manner of the invention and various modifications and embodiments can be made by those skilled in the art. in the light of the invention, without departing from the spirit of the invention.

We claim:

1. 6-(D-2-amino-2-phenylacetamido)penicillanic ac id-2,2,2-trichloro-1,1-ethanediol complex. 

1. 6-(D-2-AMINO-2-PHENYLACETAMIDO)PENCILLANIC ACID2,2,2,-TRICHLORO-1,1-ETHANEDIOL COMPLEX. 